Optically active pyridine derivative and a medicament containing the same

ABSTRACT

An optically active (−)-7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]α-azin-2-one of the formula (I) or salt thereof. The compound has an excellent anti-inflammatory activity, and other biological activity.

DETAILED DESCRIPTION OF INVENTION

1. Technical Field

The present invention relates to(−)-7-[2-cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one,a salt thereof, pharmaceutical preparations containing them.(−)-7-[2-(Cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-oneof the present invention inhibits IκB kinase β (IKK-β or IKK-beta)activity, thus inhibit nuclear factor kappa B (NF-κB) activity, and canbe used for the prophylaxis and treatment of diseases associated withNF-κB activity, in particular for the treatment of inflammatorydiseases.

2. Background Art

Nuclear factor kappa B (NF-κB) belongs to a family of closely relatedhomo- and hetero-dimeric transcription factor complexes composed ofvarious combinations of the Rel/NF-κB family of polypeptides. NF-κB andrelated family members are involved in the regulation of more than 50genes relating to immune and inflammatory responses ((Barnes P J, KarinM (1997) N Engl J Med 336, 1066-1071) and (Baeuerle P A, Baichwal V R(1997) Adv Immunol 65, 111-137)). In most cell types, NF-κB is presentas a heterodimer comprising a 50 kDa and a 65 kDa subunit (p50/RelA).The heterodimer is sequestered in the cytoplasm in association withinhibitor of NF-κB (IκB)-family of proteins to be kept in an inactivestate. IκB-family proteins mask the nuclear translocation signal ofNF-κB. Upon stimulation of cells with various cytokines (e.g. TNF-α,IL-1), CD40 ligand, lipopolysaccharide (LPS), oxidants, mitogens (e.g.phorbol ester), viruses or many others. IκB proteins are phosphorylatedat specific serine residues, poly-ubiquitinated, and then degradedthrough a proteasome-dependent pathway. Freed from IκB, the active NF-κBis able to translocate to the nucleus where it binds in a selectivemanner to preferred gene-specific enhancer sequences. Among the genesbeing regulated by NF-κB are many coding for pro-inflammatory mediators,cytokines, cell adhesion molecules, and acute phase proteins. Expressionof several of these cytokines and mediators in turn can lead to furtheractivation of NF-κB via autocrine and paracrine mechanisms.

Broad evidence is available that suggests a central role of NF-κB inmany inflammatory disorders including airway inflammation and asthma((Yang L et al., J Exp Med 188 (1998), 1739-1750), (Hart L A et al. Am JRespir Crit Care Med 158 (1998), 1585-1592), (Stacey M A et al., BiochemBiophys Res Commun 236 (1997), 522-526) (Barnes P and Adcock I M, TrendsPharmacol Sci 18 (1997), 46-50)).

Further, it has been shown that glucocorticoids, which are by far themost effective treatment for asthma, inhibit airway inflammation bydirectly interacting with and inhibiting the activity of thetranscription factors NF-κB and activating peptide-1 (AP-1) ((Barnes P(1997) Pulmon Pharmacol Therapeut 10, 3-19) and (Dumont A et al. (1998)Trends Biochem Sci 23, 233-235)).

In general, inhibition of NF-κB activation results in stronganti-inflammatory effects similar or superior to those brought upon bysteroids. Consequently, NF-κB inhibition should improve inflammatorysymptoms typical for asthma; allergic rhinitis; atopic dermatitis;hives; conjunctivitis; vernal catarrh; rheumatoid arthritis; systemiclupus erythematosus; psoriasis; diabrotic colitis; systemic inflammatoryresponse syndrome; sepsis; polymyositis; dermatomyositis; Polyaritisnodoa; mixed connective tissue disease; Sjoegren's syndrome; gout, andthe like.

Further, several studies imply that NF-κB plays an essential role inneoplastic transformation. For example, NF-κB is associated with celltransformation in vitro and in vivo as a result of gene overexpression,amplification, rearrangement, or translocation (Mercurio, F., andManning, A. M. (1999) Oncogene, 18: 6163-6171). In certain humanlymphoid tumor cells, the genes of NF-κB family members are rearrangedor amplified. Its possible involvement in cancer pathology is alsodisclosed in Mayo, M. W., Baldwin A. S. (2000) Biochmica et BiophysicaActa 1470 M55-M62. Mayo M. W. et al., discloses the inhibition of NF-κBresults in the blockage the initiation and/or progression of certaincancer, particularly colorectal cancer.

Finally, NF-κB may also be involved in the regulation of neuronal celldeath. It has been shown that NF-κB becomes activated and promotes celldeath in focal cerebral ischemia (Nature medicine Vol. 5 No. 5, May1999).

Extensive research during the past years led to the identification of anIκB kinase (IKK) complex as being responsible for the signal-induced IκBphosphorylation ((Mercurio, F., and Manning, A. M. (1999) CurrentOpinion in Cell Biology, 11: 226-232), (Mercurio, F., and Manning, A. M.(1999) Oncogene, 18: 6163-6171), (Barnkett, M., and Gilmore T. D. (1999)Oncogene 18, 6910-6924), (Zandi, E., and Karin, M., (1999) 19:4547-4551), (Israel, A., (2000) trends in CELL BIOLOGY. 10: 129-133),and (Hatada, E. N, et al. (2000) Current Opinion in Immunology, 12:52-58)). This complex is most likely the site of integration of all ofthe different stimuli leading to NF-κB activation. The IKK-complex(molecular weight 700-900 kDa) is composed of various proteins includingtwo homologous IκB kinases, called IKK-α and IKK-β, an upstream kinase,NIK which induces NF-κB, a scaffold protein called IKAP, which tetherstogether the three kinases, and a regulatory subunit IKK-γ, whichpreferentially interacts with IKK-β.

IKK-β is a 756 amino acid serine-threonine kinase showing 52% identityto and the same domain structure as IKK-α ((Mercurio F et al. (1997)Science 278, 860-866.), (Woronicz J D et al. (1997) Science 278,866-869.), (Zandi E et al. (1997) Cell 91, 243-252.). IKK-β formshomo-dimers and hetero-dimers with IKK-α in vitro and in cells,respectively. IKK-β also interacts with IKK-γ, IKAP, NIK and IκBα.Recombinant IKK-β phosphorylates IκBα and IκBβ at specific serineresidues with equal efficacy (Li J et al. (1998) J Biol Chem 273,30736-30741.), (Zandi E, Chen Y, Karin M (1998) Science 281,1360-1363.). IKK-β shows a higher constitutive kinase activity ascompared to IKK-α. This is in agreement with data suggesting thatover-expression of IKK-β activates the transcription of aNF-κB-dependent reporter gene with a higher efficacy as compared toIKK-α. IKK-β has been shown to be activated in various cell lines orfresh human cells in response to various stimuli including TNF-α, IL-1β,IL-1β, LPS, anti-CD3/anti-CD28 co-stimulation, protein kinase C andcalcineurin, B-cell receptor/CD40 ligand stimulation, and vanadate.IKK-β is activated in fibroblast-like synoviocytes (FLS) isolated fromthe synovium of patients suffering from rheumatoid arthritis orosteoarthritis (Zandi E et al. (1997) Cell 91, 243-252.), (O'Connell M Aet al. (1998) J Biol Chem 273, 30410-30414.), (Kempiak S J et al. (1999)J Immunol 162, 3176-3187.). Furthermore, IKK-β can be activated by thestructurally related upstream kinases MEKK-1 and NIK, most likelythrough phosphorylation of specific serine residues within the T-loop(activation loop) and by certain protein kinase C isoforms ((Nakano H etal. (1998) Proc Natl Acad Sci USA 95, 3537-3542.), (Lee F S et al.(1998) Proc Natl Acad Sci USA 95, 9319-9324.), (Nemoto S et al. (1998)Mol Cell Biol 18, 7336-7343.), (Lallena M J et al. (1999) Mol Cell Biol19, 2180-2188.)). A catalytically inactive mutant of IKK-β has beenshown to inhibit activation of NF-κB by TNF-α, IL-1β, LPS,anti-CD3/anti-CD28 stimulation ((Mercurio F et al. (1997) Science 278,860-866.), (Woronicz J D et al. (1997) Science 278, 866-869.)). The sameeffects are observed when MEKK1 or NIK are overexpressed. Additionally,IKK-β mutations in the activation loop inhibited IL-1 and TNF-αsignaling (Delhase M et al. (1999) Science 284, 309-313.). Based on theexperimental results described above, there is clear-cut evidence for apivotal involvement of IKK-β in various pathways leading to NF-κBactivation.

In summary, the specific inhibition of IKK-β should result in a stronganti-inflammatory and immuno-modulatory effect in vivo with thepotential of improving the underlying causes of asthma and otherdiseases. In addition, anti-tumor and anti-ischemic effects of an IKK-βinhibitor may be expected.

Manna et al., disclose 4,6-disubstituted 3-cyano-2-aminopyridinesrepresented by general formula:

wherein

-   (R″, R″) represent (OCH₃, OCH₃), (Cl, Cl), (H, Cl), (H, Br), (H,    CH₃), (H, OCH₃), (H, NO₂), or (H, N(CH₃)₂), or    as a general anti-inflammatory, analgesic, and antipyretic agent    (Eur J. Med. Chem. 34, 245-254(1999)).

Manna et al. neither disclose pyridine derivatives with aliphatic groupsat position 4 of the pyridine ring, nor suggest IKK-β kinase or NF-κBinhibitory activity on the above known pyridine derivatives.

The development of a novel compound having effective anti-inflammatoryactions based on a specific and selective inhibitory activity to IKK-βkinase has been desired.

SUMMARY OF THE INVENTION

As the result of extensive studies on chemical modification of pyridinederivatives, the present inventors have found that the compounds ofnovel chemical structure related to the present invention haveunexpectedly excellent IKK-β kinase inhibitory activity. This inventionis to provide(−)-7-[2-(cyclopropylmethoxy)-6-hydroxy-phenyl]-5-[(3S)-3-piperidinyl]-1,4-hydro-2H-pyrido[2,3-d][1,3]oxazin-2-one of the formula (I):

and the salts thereof:

The compound of the present invention is optically active andsurprisingly shows excellent IKK-β kinase inhibitory activity, cytokineinhibitory activity, and anti-inflammatory activity in vivo evenstronger than corresponding racemic modification or its enantiomer(+)-7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3R)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3d][1,3]oxazin-2-one.It is, therefore, suitable especially as a reagent to inhibit activationof NF-κB and in particular for the production of medicament or medicalcomposition, which may be useful to treat NF-κB dependent diseases.

More specifically, since(−)-7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one of the present invention inhibits IKK-β kinaseactivity, it is useful for treatment and prophylaxis of diseasesinvolving NF-κB activity as follows: inflammatory symptoms includingasthma; allergic rhinitis; atopic dermatitis, hives; conjunctivitis;vernal catarrh; chronic arthrorheumatism; systemic lupus erythematosus;psoriasis; diabrotic colitis; systemic inflammatory response syndrome(SIRS); sepsis; polymyositis; dermatomyositis (DM); Polyaritis nodoa(PN); mixed connective tissue disease (MCTD); Sjoegren's syndrome; gout;and the like.

The compound of the present invention is also useful for treatment andprophylaxis of diseases like ischemia and tumor, since the diseases alsorelate to IKK-β kinase and NF-κB activity.

The compound of the present invention can be prepared by combiningvarious known methods. In some embodiments, one or more of thesubstituents, such as amino group, carboxyl group, and hydroxyl group ofthe compounds used as starting materials or intermediates areadvantageously protected by a protecting group known to those skilled inthe art. Examples of the protecting groups are described in “ProtectiveGroups in Organic Synthesis (2^(nd) Edition)” by Greene and Wuts.

The compound of the formula (II):

(wherein —OX represents hydroxyl group or protected hydroxy group by anappropriate protecting group (e.g., benzyl, methoxybenzy, and silyl)) isreacted with an aldehyde of the formula (III):

(wherein X′ represents H or protecting group including, for instance,alkoxycarbonyl such as ethoxycarbonyl, tertiary butoxycarbonyl or thelike: or other substituents which can be easily converted to H byconventional methods)CNCH₂COOR¹  (IV)(wherein R¹ is CR¹¹R¹²R¹³, in which R¹¹, R¹² and R¹³ are eachindependently C₁₋₆alkyl or aryl),

-   and an ammonium salt such as ammonium acetate to obtain the compound    represented by the formula (V):    wherein-   X, X′, and R¹ are the same as defined above.

The reaction can be carried out without a solvent or in a solventincluding, for instance, ethers, such as dioxane, and tetrahydrofuran;aromatic hydrocarbons such as benzene, toluene and xylene; nitrites suchas acetonitrile; amides such as dimethyl-formamide (DMF) anddimethylacetamide; sulfoxides such as dimethyl sulfoxide, and others.The reaction temperature is usually, but not limited to, about 50° C. to200° C. The reaction may be conducted for, usually, 30 minutes to 48hours and preferably 1 to 24 hours. The compounds of the general formula(II), (III), (IV), and an ammonium salt such as ammonium acetate can becommercially available, or can be prepared by the use of knowntechniques.

Next, —COO—R¹ moiety of the compound (V) is converted to —CH₂—OH withthe use of conventional ester reduction method using reducing agent suchas lithium aluminum hydride, lithium borohydride, and sodium bis(2-methoxyethoxy) aluminum hydride (step1). Then the compound (VI) isconverted to the compound (VII) by conventional circulization methodsusing e.g., phosgene, diphosgene and triphosgene (step2). The protectinggroups in —OX and —NX′ in the compound (VII) can be removed by theconventional methods, e.g., acid treatment to give the compound(VIII)(step3). The chiral isomer separation of compound (VIII) gave thecompound (I). This chiral isomer separation is effected by, for example,liquid chromatografy using a chiral column consisting of opticallyactive amino acid, sugar or others, preferably with HPLC orrecrystalization method using optically active organic acid such as(−)-di-p-toluoyl-L-tertaric acid.

The compound (I) can also be obtained when chiral separation step isperformed before any of step1, step2 or step3.

Typical salts of the compound shown by the formula (I) include saltsprepared by reaction of the compound of the present invention with amineral or organic acid, or an organic or inorganic base. Such salts areknown as acid addition and base addition salts, respectively.

Acids to form acid addition salts include inorganic acids such as,without limitation, sulfuric acid, phosphoric acid, hydrochloric acid,hydrobromic acid, hydriodic acid and the like, and organic acids, suchas, without limitation, p-toluenesulfonic acid, methanesulfonic acid,oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid,citric acid, benzoic acid, acetic acid, and the like.

Base addition salts include those derived from inorganic bases, such as,without limitation, ammonium hydroxide, alkaline metal hydroxide,alkaline earth metal hydroxides, carbonates, bicarbonates, and the like,and organic bases, such as, without limitation, ethanolamine,triethylamine, tris(hydroxymethyl)aminomethane, and the like. Examplesof inorganic bases include, sodium hydroxide, potassium hydroxide,potassium carbonate, sodium carbonate, sodium bicarbonate, potassiumbicarbonate, calcium hydroxide, calcium carbonate, and the like.

The compound of the present invention or a salts thereof, may bemodified to form lower alkylesters or known other esters; and/orhydrates or other solvates. Those esters, hydrates, and solvates areincluded in the scope of the present invention.

The compound of the present invention may be administered in oral forms,such as, without limitation normal and enteric coated tablets, capsules,pills, powders, granules, elixirs, tinctures, solution, suspensions,syrups, solid and liquid aerosols and emulsions. They may also beadministered in parenteral forms, such as, without limitation,intravenous, intraperitoneal, subcutaneous, intramuscular, and the likeforms, well known to those of ordinary skill in the pharmaceutical arts.The compounds of the present invention can be administered in intranasalform via topical use of suitable intranasal vehicles, or via transdermalroutes, using transdermal delivery systems well known to those ofordinary skilled in the art.

The dosage regimen with the use of the compound of the present inventionis selected by one of ordinary skill in the arts, in view of a varietyof factors, including, without limitation, age, weight, sex, and medicalcondition of the recipient, the severity of the condition to be treated,the route of administration, the level of metabolic and excretoryfunction of the recipient, the dosage form employed, the particularcompound and salt thereof employed.

The compound of the present invention is preferably formulated prior toadministration together with one or more pharmaceutically acceptableexcipients. Excipients are inert substances such as, without limitationcarriers, diluents, flavoring agents, sweeteners, lubricants,solubilizers, suspending agents, binders, tablet disintegrating agentsand encapsulating material.

Yet, another embodiment of the present invention is pharmaceuticalformulation comprising the compound of the invention and one or morepharmaceutically acceptable excipients that are compatible with theother ingredients of the formulation and not deleterious to therecipient thereof. Pharmaceutical formulations of the invention areprepared by combining a therapeutically effective amount of thecompounds of the invention together with one or more pharmaceuticallyacceptable excipients therefor. In making the compositions of thepresent invention, the active ingredient may be mixed with a diluent, orenclosed within a carrier, which may be in the form of a capsule,sachet, paper, or other container. The carrier may serve as a diluent,which may be solid, semi-solid, or liquid material which acts as avehicle, or can be in the form of tablets, pills powders, lozenges,elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments,containing, for example, up to 10% by weight of the active compound,soft and hard gelatin capsules, suppositories, sterile injectablesolutions and sterile packaged powders.

For oral administration, the active ingredient may be combined with anoral, and non-toxic, pharmaceutically-acceptable carrier, such as,without limitation, lactose, starch, sucrose, glucose, sodium carbonate,mannitol, sorbitol, calcium carbonate, calcium phosphate, calciumsulfate, methyl cellulose, and the like; together with, optionally,disintegrating agents, such as, without limitation, maize, starch,methyl cellulose, agar bentonite, xanthan gum, alginic acid, and thelike; and optionally, binding agents, for example, without limitation,gelatin, acacia, natural sugars, beta-lactose, corn sweeteners, naturaland synthetic gums, acacia, tragacanth, sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes, and the like; and,optionally, lubricating agents, for example, without limitation,magnesium stearate, sodium stearate, stearic acid, sodium oleate, sodiumbenzoate, sodium acetate, sodium chloride, talc, and the like.

In powder forms, the carrier may be a finely divided solid, which is inadmixture with the finely divided active ingredient. The activeingredient may be mixed with a carrier having binding properties insuitable proportions and compacted in the shape and size desired toproduce tablets. The powders and tablets preferably contain from about 1to about 99 weight percent of the active ingredient which is the novelcomposition of the present invention. Suitable solid carriers aremagnesium carboxymethyl cellulose, low melting waxes, and cocoa butter.

Sterile liquid formulations include suspensions, emulsions, syrups andelixirs. The active ingredient can be dissolved or suspended in apharmaceutically acceptable carrier, such as sterile water, sterileorganic solvent, or a mixture of both sterile water and sterile organicsolvent.

The active ingredient can also be dissolved in a suitable organicsolvent, for example, aqueous propylene glycol. Other compositions canbe made by dispersing the finely divided active ingredient in aqueousstarch or sodium carboxymethyl cellulose solution or in suitable oil.

The formulation may be in unit dosage form, which is a physicallydiscrete unit containing a unit dose, suitable for administration inhuman or other mammals. A unit dosage form can be a capsule or tablets,or a number of capsules or tablets. A “unit dose” is a predeterminedquantity of the active compound of the present invention, calculated toproduce the desired therapeutic effect, in association with one or moreexcipients. The quantity of active ingredient in a unit dose may bevaried or adjusted from about 0.1 to about 1000 milligrams or moreaccording to the particular treatment involved.

Typical oral dosages of the present invention, when used for theindicated effects, will range from about 0.01 mg/kg/day to about 100mg/kg/day, preferably from 0.1 mg/kg/day to 30 mg/kg/day, and mostpreferably from about 0.5 mg/kg/day to about 10 mg/kg/day. In the caseof parenteral administration, it has generally proven advantageous toadminister quantities of about 0.001 to 100 mg/kg/day, preferably from0.01 mg/kg/day to 1 mg/kg/day. The compounds of the present inventionmay be administered in a single daily dose, or the total daily dose maybe administered in divided doses, two, three, or more times per day.Where delivery is via transdermal forms, of course, administration iscontinuous.

The effect of the present compound was examined by the following assaysand pharmacological tests.

[IKK-β Kinase Inhibitory Assay]

(1) Preparation of IKK-β Kinase Protein

A cDNA fragment encoding human IKK-β open reading frame was generated byPCR with the use of a pair of primers designed from the publishedsequence (Woronicz J D et al. (1997) Science 278, 866-869). A templatewas obtained from Quickclone cDNA (Clontech) using Elongase™Amplification kit (Life Technologies). The DNA fragments generated byPCR were gel-purified and subcloned into pBluescript. The cDNA fragmentcloned in pBluescript was inserted into pcDNA3.1/His C KpnI/NotI, andtransferred into pVL1393 SmaI/XbaI (Pharmingen) to construct abaculovirus transfer vector. Then the vector, together with thelinearized baculovirus (BaculoGold™, Pharmingen) was used to transfectSf21 cells (Invitrogen, San Diego, Calif.). Generated recombinantbaculovirus was cloned and amplified in Sf21 cells, grown in TNM-FHinsect cell medium (Life Technologies, Inc.) supplemented with 10% FCS,50 g/ml Gentamycin, 0.1% Pluronic F68 (Life Technologies, Inc.) assuspension culture (200 ml in 1 L Erlenmeyer flask; 27° C.; 130 rpm).Sf21 cells were infected with this amplified virus with a multiplicityof infection of 5 following standard protocols (Crossen R, Gruenwald S(1997) Baculovirus Expression Vector System Instruction Manual,Pharmingen Corporation) and harvested 48 hrs later. The cells were lysedto obtain the produced chimeric protein of IKK-β kinase fused byhistidine (His-tagged IKK-beta).

(2) The Preparation of Purified GST-IκBα Fusion Proteins

An expression vector containing the nucleotide sequence encoding fusionprotein of GST with amino acid residues 1 to 54 of IκBα under thecontrol of an IPTG-inducible promoter was constructed. The expressionvector was introduced in E. coli and the transformant was cultured andlysed to obtain a GST-IκBα fusion protein. Then the resulting GST-IκBαfusion protein was purified and biotinated for kinase assay.

(3) The Measurement of IKK-β Kinase Activity

The 96-well format kinase assay of IKK-β were performed to test theinhibitory activity of the compounds of the present invention. First, 5μl of a test compound was put in the presence of 2.5% dimethyl sulfoxide(DMSO) in each well in a U-bottomed 96-well plate (Falcon). For controlwells of background (BG) and total phosphorylation (TP), 5 μl of 2.5%DMSO was put. Recombinant IKK-β (final 0.6 μg/ml) and bio-GST-IκBα(1-54) (final 0.2 μM) were diluted in 25 μl of 2× kinase buffer 0 (40 mMTris-HCl, pH 7.6, 40 mM MgCl₂, 40 mM O-glycerophosphate, 40 mMp-nitro-phenylphosphate, 2 mM EDTA, 40 mM creatine phosphate, 2 mM DTT,2 mM Na₃VO₄, 0.2 mg/ml BSA and 0.8 mM phenylmethylsulfonyl fluoride) andtransferred to the 96-well plate. Bio-GST-IκBα (1-54) in 25 μl of 2×kinase buffer β without IKK-β was transferred to BG wells. Then 20 μl of12.5 μM ATP, 62.5 μCi/ml [β-³³P] ATP (Amersham Pharmacia Biotech) wasadded and the resulting mixture was incubated for 2 hrs at roomtemperature. The kinase reactions were terminated by the addition of 150μl of termination buffer (100 mM EDTA, 1 mg/ml BSA, 0.2 mg NaN₃). Onehandred and fifty μl of the sample were transferred to astreptavidin-coated, white MTP (Steffens Biotechniche Analysen GmbH#08114E14.FWD) to capture the biotinylated substrates. After 1 hr ofincubation, non-bound radioactivity was eliminated by washing the wellsfive times with 300 μl of washing buffer including 0.9% NaCl and 0.1%(w/v) Tween-20 with the use of a MW-96 plate washer (BioTec). The boundradioactivity was determined after the addition of 170 μl MicroScint-PSscintillation cocktail (Packard) using a TopCount scintillation counter.

[Syk Tyrosine Kinase Inhibitory Assay for Selectivity]

(1) Preparation of Syk Protein

A cDNA fragment encoding human Syk openreading frame was cloned fromtotal RNA of human Burkitt's lymphoma B cell lines, Raji (American TypeCulture Collection), with the use of RT-PCR method. The cDNA fragmentwas inserted into pAcG2T (Pharmingen, San Diego, Calif.) to construct abaculovirus transfer vector. Then the vector, together with thelinearized baculovirus (BaculoGold™, Pharmingen), was used to transfectSf21 cells (Invitrogen, San Diego, Calif.).

Generated recombinant baculovirus was cloned and amplified in Sf21cells. Sf21 cells were infected with this amplified high titer virus toproduce a chimeric protein of Syk kinase fused byglutathione-S-transferase (GST).

The resulting GST-Syk was purified with the use of glutathione column(Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to themanufacturer's instruction. The purity of the protein was confirmed tobe more than 90% by SDS-PAGE.

(2) Synthesize of a Peptide

Next, a peptide fragment of 30 residues including two tyrosine residues,KISDFGLSKALRADENYYKAQTHGKWPVKW, was synthesized by a peptidesynthesizer. The N-terminal of the fragment was then biotinylated toobtain biotinylated activation loop peptide (AL).

(3) The Measurement of Syk Tyrosine Kinase Activity

All reagents were diluted with the Syk kinase assay buffer (50 mMTris-HCl (pH 8.0), 10 mM MgCl₂, 0.1 mM Na₃VO₄, 0.1% BSA, 1 mM DTT).First, a mixture (35 μl) including 3.2 μg of GST-Syk and 0.5 μg of ALwas put in each well in 96-well plates. Then 5 μd of a test compound inthe presence of 2.5% dimethyl sulfoxide (DMSO) was added to each well.To this mixture was added 300 μM ATP (10 μl) to initiate the kinasereaction. The final reaction mixture (50 μl) consists of 0.65 nMGST-Syk, 3 μM AL, 30 μM ATP, a test compound, 0.25% DMSO, and a Sykkinase assay buffer.

The mixture was incubated for 1 hr at room temperature (RT), and thereaction was terminated by the addition of 120 μl of termination buffer(50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 500 mM NaCl, 0.1% BSA). Themixture was transferred to streptavidin-coated plates and incubated for30 min. at room temperature to combine biotin-AL to the plates. Afterwashing the plates with Tris-buffered saline (TBS) (50 mM Tris-HCl (pH8.0), 138 mM NaCl, 2.7 mM KCl) containing 0.05% Tween-20 for 3 times,100 μl of antibody solution consisting of 50 mM Tris-HCl (pH 8.0), 138mM NaCl, 2.7 mM KCl, 1% BSA, 60 ng/ml anti-phosphotyrosine monoclonalantibody, 4G10 (Upstate Biotechnology), which was labeled with europiumby Amersham Pharmacia's kit in advance, was added and incubated at roomtemperature for 60 minutes. After washing, 100 μl of enhancementsolution (Amersham Pharmacia Biotech) was added and then time-resolvedfluorescence was measured by multi-label counter ARVO (Wallac Oy,Finland) at 340 nm for excitation and 615 nm for emission with 400 msecof delay and 400 msec of window.

[The Measurement of RANTES Production in Response to TNF-α from A549cells]

(1) Preparation of A549 Cells

The A549 human lung epithelium cell line (ATCC #CCL-885) was maintainedin Dulbecco's modified Eagle's medium (D-MEM, Nikken BiomedicalInstitute) supplemented with 10% FCS (Gibco), 100 U/ml penicillin, 100μg/ml streptomycin, and 2 mM glutamine (culture medium). Forty thousand(4×10⁴) cells (80 μl/well) were seeded in each well of 96 wellflat-bottom tissue culture plate (Falcon #3072). The plate was allowedto stand for 2 hrs, thus the cells were adhered to the bottom of eachwell. To the each well was added 10 μl vehicle (1% DMSO), serialdilutions of test compounds in 1% DMSO, or 5 nM Dexamethasone in 1% DMSOas a reference. The mixture (90 μl/well) was incubated for 1 hr at 37°C. After 1 hr, 1 μg/ml TNF-α (10 μl) in culture medium was added to themixture to obtain 100 μl of reaction mixture. The reaction mixture wascultured for 24 hrs to stimulate the cells with 100 ng/ml TNF-α. Cellswith vehicle without TNF-α stimulation were also prepared.

(2) Measurement of RANTES Production

Then the concentration of RANTES released from the cells in thesupernatants of each well was determined using a quantitative sandwichenzyme immunoassay technique. First, 2 μg/ml mouse anti-huRANTES mAb(R&D Systems, #mAb678) in PBS buffer (pH 7.4, 1001 μl) was put in eachwell of 96-well NUNC fluoro plate (Nalge Nunc, New York USA) (Final 200ng/well) and the plate was allowed to stand for overnight at 4° C. to becoated by the antibody. Each well of the plate was then washed with 350μl wash buffer (0.05% Tween-20, 0.85% NaCl, and 25 mM Tris/HCl pH7.4)for three times. Blocking buffer containing 1% BSA (Sigma 99% pure, 100g), 5% sucrose (Nacalai tesque, 99% pure, 500 g), and 0.02% azide(Nacalai tesque, 100%, 500 g) were added (200 μl) to each well and thenthe plate was allowed to stand for 4 hours to stabilize the coatedantibody. Next, 50 μl supernatants of cell culture prepared in (1) abovewere put in each well of the 96-well NUNC fluoro plate with coatedantibody. Recombinant Human RANTES (Pepro Tech, Inc. #300-06) was usedas the standard for the determination of RANTES production (linear rangebetween 1 and 10 ng/ml). Eu-labelled mouse anti-huRANES mAb (60 ng/ml:R&D Systems, #mAb278) in PBS supplemented by 1% BSA and 0.05% Tween 20was added (50 μl) to each well. The reaction mixtures were incubated atroom temperature for 4 hrs. After washing with wash buffer (0.05%Tween-20, 0.85% NaCl, and 25 mM Tris/HCl pH7.4, 350 μl/well) for 5 timeswith the use of a Sera Washer (Bio-Tech, #MW-96R), the enhancementsolution (DELFIA, #1244-405, 100 μl/well) was added to each well. Theplate was incubated for 10 minutes at room temperature with moderateshaking. Fluorescent intensity was measured using a DELFIA fluorimeter(Wallac). Excitation was performed at 340 nm and emission was measuredat 615 nm.

[The Measurement of TNF-α Production in Response to LPS from PeripheralBlood Mononuclear Cells (PBMC)]

(1) Preparation of PBMC

Human PBMC were prepared by first obtaining blood from healthy donorsand isolating the cells from the blood. The isolation was done by Ficollgradient-centrifugation method using Ficoll Pacque (Pharmacia#17-1440-02). Within three hours from donation, the isolated PBMC wasused. After three times washing with PBS, PBMC were resuspended withRPMI 1640 (Nikken BioMedical Institute) supplemented with 10% FCS(Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine(culture medium). The cells (1×10⁵ in 150 μl/well) were seeded in eachwell of 96 well flat-bottom tissue culture plate (Falcon #3072). To theeach well was added 20 μl vehicle (1% DMSO), serial dilutions of testcompounds in 1% DMSO, or 250 nM Dexamethasone in 1% DMSO as a reference.The mixture (170 μl/well) was incubated for 1 hr at 37° C. After 1 hr,20 ng/ml LPS (30 μl) in culture medium was added to the mixture toobtain 200 μl of reaction mixture. The reaction mixture was cultured for7 hrs to stimulate the cells with 3 ng/ml LPS. Cells with vehiclewithout LPS stimulation were also prepared. The supernatants of thereaction mixture were then collected.

(2) Measurement of TNF-α Production

The TNF-α concentration in the supernatants was determined using aDuoSet™ ELISA Development Kit (GenzymeTechne, Minneapolis, USA)following the manufacturer's recommendations. First, 4 μg/ml of mouseanti-human TNF-α Ab in PBS buffer (100 μl) was put in each well of96-well plate (NUNC, Maxisorp™) and the plate was allowed to stand forovernight at 4° C. to be coated with the antibody. Each well of theplate was then washed 5 times with 350 μl of wash buffer containing PBS,0.05% Tween 20 (Nakalai tesque) using Sera Washer (Bio-Tech, #MW-96R).To each well was added 300 μl of 1% BSA (Sigma), 5% sucrose in PBS.After 2 hrs incubation at room temperature, the buffer was discarded,and 50 μl of culture medium was added. Next, 50 μl supernatant ofstimulated cell culture prepared (1) above was put in each well of the96-well plate. Recombinant human TNF-α (Genzyme Techne) was used as thestandard for the determination of TNF-α production (linear range between30 and 2,000 pg/ml). The reaction mixtures were incubated for 1 hr atroom temperature. After 5 times washing, 100 μl biotinylated goatanti-human TNF-α antibody (Genzyme Techne, 300 ng/ml) in 0.1% BSA, 0.05%Tween in PBS (Reagent diluent) was added to each well, and incubated atroom temperature for 1 hr. After 5 times washing, 100 μl ofStreptavidin-conjugated horseradishperoxidase (Genzyme Techne, 1/100 inReagent diluent) was added to each well. After 20 min, each well of theplate was washed 5 times with wash buffer (350 μl/well). The substrateof hourseradishperoxidase and H₂O₂ (TMBZ peroxidase detection kit,SUMILON #ML-1120T) were added to the mixture and the mixture was allowedto stand at room temperature. The reaction was terminated after 10 minby adding 2N H₂SO₄. Optical density at 450 nm was measured with the useof a microplate reader (Labosystems, Multiscan Multisoft).Quantification of TNF-α production in each sample was performed bycomparison of optical densities between each sample and the standardcurve.

[The Measurement of IL-2 Production in Jurkat T Cells in Response toAntibody Stimulation]

IL-2 production was measured in Jurkat T cells (E6-1 clone; ATCC #TIB-152) in response to stimulation with anti-CD3/anti-CD28 antibodies.

(1) Preparation of Immobilized Antibodies

First, anti-CD3 antibodies (400 ng/well Nichirei, NU-T3 4 μg/ml in 100μl Dulbecco's PBS) were put in each well of 96-well plate (Falcon #3072)and the plate was allowed to stand for 2 hrs at room temperature to becoated with the antibody. Each well of the plate was then washed with250 μl PBS 3 times.

(2) Preparation of Jurkat Cell Culture

Jurkat T cells were cultured in RPMI 1640 medium supplemented with 10%heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillinG, and 100 μg/ml streptomycin (culture medium). Two hundred thousand(2×10⁵) cells (190 μl/well) were seeded in each well of 96-well U-bottomtissue culture plates (Falcon #3077). To each well was added 10 μlvehicle (0.2% DMSO), serial dilution of compounds in 0.2% DMSO, or 25 nMcyclosporin A as a reference in 0.2% DMSO. The mixture (200 μl) wasincubated for one hour at 37° C. in a humidified 5% CO₂ environment.

(3) Stimulation of the Cell

The reaction mixture obtained in (2) (100 μl) was put in the each wellof the antibody-immobilized plate prepared in (1). To this well wasadded anti-CD28 antibodies (Nichirei, KOLT-2, 6 μg/ml in cell culturemedium, 50 μl/well) and 2.5 μg/ml goat anti-mouse kappa chain antibodies(Bethyl Laboratories, (Cat#A90-119A) 10 μg/ml in culture medium, 50μl/well). The reaction mixture in each well was incubated for 24 hrs at37° C. to stimulate cells with immobilized anti-CD3 antibodies (400ng/well) and anti-CD28 antibodies (1.5 μg/ml), and then to cross-linkreceptors on the cells with anti-mouse kappa chain antibodies (2.5μg/ml).

(4) Measurement of IL-2 Production

The supernatants of the reaction mixture were then collected. The IL-2concentration in the supernatants was determined using a DuoSet™ ELISADevelopment Kit (GenzymeTechne, Minneapolis, USA) following themanufacturer's recommendations. First, 2 μg/ml of mouse anti-huIL-2 Abin PBS buffer (100 μl) was put in each well of 96-well plate (NUNC,Maxisorp™) and the plate was allowed to stand for overnight at 4° C. tobe coated with the antibody. Each well of the plate was then washed 5times with 350 μl of wash buffer containing PBS, 0.05% Tween 20 (Nakalaitesque) using Sera Washer (Bio-Tech, #MW-96R). To each well was added250 μl of 1% BSA (Sigma) in PBS, 0.05% Tween 20 (dilution buffer). After2 hrs incubation at room temperature, the buffer was discarded, and 50μl of culture medium was added. Next, 50 μl supernatant of stimulatedcell culture prepared (3) above was put in each well of the 96-wellplate with coated mouse anti-huIL-2 antibody. Recombinant Human IL-2(Genzyme Techne) was used as the standard for the determination of IL-2production (linear range between 200 and 5,400 pg/ml). The reactionmixtures were incubated for 1 hr at room temperature. After 5 timeswashing, 100 μl biotinylated rabbit anti-huIL-2 antibody (GenzymeTechne, 1.25 μg/ml) in dilution buffer was added to each well, andincubated at room temperature for 1 hr. After 5 times washing, 100 μl ofStreptavidin-conjugated horseradishperoxidase (Genzyme Techne, 1/1000 indilution buffer) was added to each well. After 20 min, each well of theplate was washed 5 times with wash buffer (350 μl/well). Substrate andH₂O₂ (TMBZ peroxidase detection kit, SUMILON #ML-1120T) were added tothe mixture and the mixture was allowed to stand at room temperature.The reaction was terminated after 10 min by adding 2N H₂SO₄. Opticaldensity at 450 nm was measured with the use of a microplate reader(Labosystems, Multiscan Multisoft). Quantification of IL-2 production ineach sample was performed by comparison of optical densities betweeneach sample and the standard curve.

[Mouse LPS-Induced TNF-α Production]

Eight weeks old BALB/c female mice were placed into two groups, acontrol group and a treated group. A solution containing 200 μg/mouse ofLPS in 0.9% physiological salt was administered by intraperitoneal (ip)injection into the control mice. Mice in the treated group were firstinjected ip with compounds of the present invention 30 minutes prior tothe LPS injection. Under anesthesia with pentobarbital (80 mg/kg, i.p.),blood was collected from the posterior venous cavity of the treated andcontrol mice at 90 min post-LPS injection into 96-well plate containing2% EDTA solution. The plasma was separated by centrifugation at 1800 rpmfor 10 minutes at 4° C. and then diluted with four times volumes ofphosphate buffer saline (pH 7.4) containing 1% bovine serum albumin.TNF-α concentration in the sample was determined using an ELISA kit(Pharmingen, San Diego, Calif.)

The mean TNF-α level in 5 mice from each group was determined and thepercent reduction in TNF-α levels was calculated. The treated miceshowed significant decrease in the level of TNF-α as compared to thecontrol mice. The result indicates that the compounds of the presentinvention can restrain LPS-induced cytokine activity.

[Rat LPS-Induced TNF-α Production]

Seven weeks old Wistar female rats were used. One mg of LPS(lypopolysaccharide) dissolved in phosphate buffer saline (pH 7.4) wasadministered intraperitoneally (i.p.) to rats. Compounds were givenorally 60 minutes prior to the LPS injection. Under anesthesia withpentobarbital (80 mg/kg, i.p.), blood was collected from the posteriorvenous cavity of the rats 120 minutes post-LPS injection and added into96-well plate containing 2% EDTA solution. The plasma was separated bycentrifugation at 1800 rpm for 10 minutes at 4° C. and then diluted withfour times volumes of phosphate buffer saline (H 7.4) containing 1%bovine serum albumin. TNF-α concentration in the sample was determinedusing an ELISA kit (Endogen, Boston, Mass.).

The mean TNF-α level in 7-8 rats from each group was determined and thepercent reduction in TNF-α levels was calculated. The treated rats,given the compounds, showed significant decrease in the level of TNF-αas compared to the control rats. The result indicates that the compoundsof the present invention can restrain LPS-induced cytokine activity.

Results of in vitro test are shown in Examples below. The datacorresponds to the compounds as yielded by solid phase synthesis andthus to levels of purity of about 40 to 90%. The compound of the presentinvention also shows excellent selectivity and strong activity incellular assays and in vivo assays. More concretely, the compound of thepresent invention shows cellular activity twice as strong ascorresponding racemic modification. Also, the compound of the presentinvention shows anti-inflammatory activity twice as strong ascorresponding racemic modification in Rat. Further, the compound wasconfirmed to be non-mutagenic according to the Ames-Test Screening.

EXAMPLES

The present invention will be described in detail below in the form ofexamples, but they should by no means be construed as defining the metesand bounds of the present invention. In the examples below, allquantitative data, if not stated otherwise, relate to percentages byweight. Mass spectra were obtained using electrospray (ES) ionizationtechniques (micromass Platform LC). Melting points are uncorrected.Liquid Chromatography-Mass spectroscopy (LC-MS) data were recorded on aMicromass Platform LC with Shimadzu Phenomenex ODS column (4.6 mm×30 mm)flushing a mixture of acetonitrile-water (9:1 to 1:9) at 1 ml/min of theflow rate. TLC was performed on a precoated silica gel plate (Mercksilica gel 60 F-254). Silica gel (WAKO-gel C-200 (75-150 elm)) was usedfor all column chromatography separations. All chemicals were reagentgrade and were purchased from Sigma-Aldrich, Wako pure chemicalindustries, Ltd., Tokyo kasei kogyo co. Ltd.

Proton nuclear magnetic resonance (1H NMR) spectra were recorded ateither 300 or 500 MHz by Bruker DRX-300. 500 Bruker UltraShield™ andchemical shifts are reported in parts per million relative totetramethylsilane (TMS).

Example 1 1-{2-[(cyclopropylmethyl)oxy]-hydroxyphenyl}ethanone

To a stirred solution of 1-(2,6-dihydroxyphenyl)ethanone (50.0 g, 328mmol) in acetone (1000 mL) were added potassium carbonate (227 g, 1643mmol) and (bromomethyl)cyclopropane (35.1 mL, 361 mmol). The mixture wasstirred at 50° C. for 2 days. The reaction mixture was filtrated onCelite®, and then the filtrate was concentrated under reduced pressure.The residue was diluted with water and extracted with ethyl acetate. Theseparated organic phase was washed with water and brine, dried overMgSO₄, filtered and concentrated under reduced pressure. The residue wassuspended in hexane. Then the suspension was stirred at 80° C. for 30min. The solution was filtered and the filtrate was allowed to cool toroom temperature. The resulting white solid was collected by filtration,washed with hexane, and dried under reduced pressure to give1-{2-[(cyclopropylmethyl)oxy]-6-hydroxyphenyl} ethanone as a pale yellowsolid (56.3 g, yield; 83%).

1-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}ethanone

To a stirred solution of1-{2-[(cyclopropylmethyl)oxy]-6-hydroxyphenyl}ethanone (56.3 g, 272mmol) in acetone (1000 mL) were added potassium carbonate (188 g, 1364mmol), 4-methoxybenzyl chloride (40.9 mL, 300 mmol) andtetrabutyl-ammonium iodide (20.2 g, 54.6 mmol). The mixture was stirredat reflux overnight. The reaction mixture was allowed to cool to roomtemperature, filtered on Celite®, and then the filtrate was concentratedunder reduced pressure. The residue was diluted with water and extractedwith ethyl acetate. The separated organic phase was washed with brine,dried over MgSO₄, filtered and concentrated under reduced pressure. Thenthe resulting white solid was recrystallized from ethanol, collected byfiltration, washed with ethanol, and dried under reduced pressure togive 1-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}ethanoneas a white solid (79.2 g, yield; 89%).

tert-butyl2-amino-4-[1-(tert-butoxycarbonyl)-3-piperidinyl]-6-{2-(cyclopropyl-methoxy)-6-[(4-methoxybenzyl)oxy]phenyl}nicotinate

A mixture of1-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}ethanone (10.00g, 30.638 mmol), tert-butyl 3-formyl-1-piperidinecarboxylate (13.069 g,61.275 mmol), tert-butylcyanoacetate (8.650 g, 61.275 mmol), andammonium acetate (6.902 g, 91.913 mmol) in dioxane (10 mL) was stirredat 90° C. overnight. After cooled to room temperature, the reactionmixture was diluted with ethyl acetate (100 mL). To the mixture wasadded chloranil (1.507 g, 6.128 mmol), and stirred at room temperature.After 1.5 hrs, ascorbic acid (1.079 g, 6.128 mmol) was added to themixture. After stirred for 1.5 hrs, the mixture was partitioned betweenethyl acetate and water. The organic phase was washed with brine, driedover MgSO₄, filtered, and then concentrated under reduced pressure. Theresulting residue was purified by column chromatography on Silica-gel(hexane/ethyl acetate=2/1) to give tert-butyl2-amino-4-[1-(tert-butoxycarbonyl)-3-piperidinyl]-6-{2-(cyclopropyl-methoxy)-6-[(4-methoxybenzyl)oxy]phenyl}nicotinateas a pale brown form (4.9 g, 24%).

tert-butyl3-[2-amino-6-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]-phenyl}-3-(hydroxymethyl)-4-pyridinyl]-1-piperidinecarboxylate

To a cooled solution of tert-butyl2-amino-4-[1-(tert-butoxycarbonyl)-3-piperidinyl]-6-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}nicotinate(4.9 g, 7.426 mmol) in tetrahydrofuran (60 mL) was added dropwiseVitride® (10 mL) under an argon atmosphere. The stirring was continuedat 0° C. for 1 hr. After quenched by saturated aqueous NH₄Cl solution,saturated aqueous potassium sodium tartrate was added to the mixture,then the mixture was stirred vigorously. The mixture was extracted withethyl acetate, washed with water and brine, dried over MgSO₄, filtered,and concentrated under reduced pressure to give tert-butyl3-[2-amino-6-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}-3-(hydroxymethyl)-4-pyridinyl]-1-piperidinecarboxylate,which was used for the next step without further purification (4.38 g,yield; quant.).

tert-butyl3-(7-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}-2-oxo-1,4-dihydro-2H-pyrido[2,3-d[1,3]oxazin-5-yl)-1-piperidinecarboxylate

To a cooled (0° C.) solution of tert-butyl3-[2-amino-6-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}-3-(hydroxymethyl)-4-pyridinyl]-1-piperidine-carboxylate(5.0 g, 8.478 mmol), which was obtained in the step (2) of Example 17-1,and diisopropylethyl amine (4.12 mL, 25.435 mmol) in tetrahydrofuran(200 mL) under argon atmosphere was added dropwise to a solution oftriphosgene (1.258 g, 4.239 mmol) in tetrahydrofuran (100 mL). Themixture was allowed to warm to room temperature, and the stirring wascontinued for 3 hrs. After quenched by water, the mixture was extractedwith ethyl acetate. The separated organic phase was washed with brine,dried over MgSO₄, filtered, and concentrated under reduced pressure. Theresulting residue was purified by column chromatography on Silica-gel(hexane/ethyl acetate=1/1) to give tert-butyl3-(7-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}-2-oxo-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-5-yl)-1-piperidinecarboxylateas a white form (3.2 g, yield; 61%).

7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-(3-piperidinyl)-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-onehydrochloride

To a solution of tert-butyl3-(7-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}-2-oxo-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-5-yl)-1-piperidine-carboxylate(2.0 g, 3.248 mmol) in dioxane (15 mL) was added 4N HCl in dioxane (30mL) at room temperature. The stirring was continued for 3 hrs. After thesolvent was removed by evaporation, the resulting solid was trituratedwith acetonitrile, collected by filtration, and washed withacetonitrile. The solid was dried under reduced pressure to give7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-(3-piperidinyl)-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-onehydrochloride as a white solid (0.865 g, yield; 62%).

(−)-7-[2-cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-pipeidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one(−)-di-p-toluoyl-L-tertaric acid salt

A mixture of7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-(3-piperidinyl)1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one (0.700 g, 1.770 mmol) and(−)-di-p-toluoyl-L-tertaric acid (0.684 g, 0.213 mmol) was dissolved ina mixture of ethanol (30 mL) and water (3.0 mL) by heating. The mixturewas allowed to cool to room temperature and stand overnight. Theresulting precipitate was collected by filtration and washed withethanol (85% ee). The product was again recrystallized from the samesolvent (10% water/ethanol) and dried under reduced pressure to give(−)-7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one(−)-di-p-toluoyl-L-tertaric acid salt (0.115 g, >98% ee, yield; 8%).

Example 2 tert-butyl(3S)-(−)-3-(7-(2-(cyclopropylmethoxy)-6-((4-methoxybenzyl)oxy]phenyl)-2-oxo-1,4-dihydro-2Hpyrido[2,3-d][1,3]oxazin-5-yl)-1-piperidinecarboxylate

The chiral separation of tert-butyl3-(7-({2-cyclopropylmethoxy)-6[(4-methoxybenzyl)oxy]phenyl}-2-oxo-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-5-yl)-1-piperidine-carboxylatewas performed using HPLC under the following conditions:Column:DaiselCHIRALPAK OD (Daicel Chemical Industries, Ltd.)

Column size: 250*20 mm ID

Eluent: hexane/isopropanol, 60/40 (vol/vol)

Flow rate: 20 ml/min

Retention time: 31 min [(+)-isomer], 45 min [(−)-isomer]

The separated (−)-isomer, tert-butyl(3S)-(−)-3-(7-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}-2-oxo-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-5-yl)

1-piperidinecarboxylate, was obtained as a colorless form.

Molecular weight: 615.73

Mass spectrometry: 616

[α]D=−23.8° (CHCl₃, c=1.035, 23° C.)

The separated (+)-isomer, tert-butyl(3R)-(+)-3-(7-{2-(cyclopropylmethoxy)-6-[(4-methoxybenzyl)oxy]phenyl}-2-oxo-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-5-yl)-1-piperidinecarboxylate,was obtained as a colorless form.

Molecular weight: 615.73

Mass spectrometry: 616

[α]_(D)=+22.50 (CHCl₃, c=1.012, 22° C.)

(−)-7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-onehydrochloride

To a solution of tert-butyl(3S)-(−)-3-(7-{2-(cyclopropylmethoxy)-6-[(4-methoxy-benzyl)oxy]phenyl}-2-oxo-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-5-yl)-1-piperidinecarboxylate(1.0 g, 1.624 mmol) in dioxane (15 mL) was added 4N HCl in dioxane (30mL) at room temperature. The stirring was continued for 3 hrs. After thesolvent was removed by evaporation, the resulting solid was trituratedwith acetonitril, collected by filtration, and washed with acetonitrile.The solid was recrystallized from methanol to give(−)-7-[2-(cyclopropylmethoxy)-6-hydroxy-phenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-onehydrochloride as a white solid (0.502 g, yield; 71%). The absoluteconfiguration for the chiral atom was determined with (S) by the X-rayanalysis.

Molecular weight: 431.92

Mass spectrometry: 396

Melting point: 260° C.

[α]_(D)=−21.1 (DMF, c=0.908, 23° C.)

¹H-NMR (500 MHz, DMSO-d6): 0.26-0.37 (2H, m), 0.51-0.63 (2H, m),1.20-1.31 (1H, m), 1.72-1.95 (4H, m), 2.80-2.96 (2H, m), 3.17-3.37 (3H,m), 3.79-3.88 (2H, m), 5.48 (1H, d, J=14.2 Hz), 5.53 (1H, d, J=14.2 Hz),6354 (2H, d, J=8.2 Hz), 7.17 (1H, t, J=8.2 Hz), 7.77 (1H, s), 8.91 (1H,br), 9.11 (1H, br), 10.96 (1H, s), 11.62 (1H, s).

IKK-beta kinase inhibitory activity: IC₅₀=4 nM

(+)-7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3R)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-onehydrochloride

According to the similar synthetic procedure above,(+)-7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3R)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-onehydrochloride was obtained as a white solid.

Molecular weight: 431.92

Mass spectrometry: 396

Melting point: 260° C.

[α]D=+21.5° (DMF, c=0.920, 25° C.)

IKK-beta kinase inhibitory activity: IC₅₀=59 nM

1. An optically active(−)-7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-oneof the formula (I):

or a salt thereof.
 2. The compound or a salt as claimed in claim 1,having an optical purity of at least 90% enantiomeric excess.
 3. Thecompound or a salt as claimed in claim 1, having an optical purity of atleast 95% enantiomeric excess.
 4. (canceled)
 5. A pharmaceuticalcomposition comprising the compound or a salt thereof as claimed inclaim 1, 2 or 3 and at least one pharmaceutically acceptable excipient.6. A pharmaceutical composition having IκB kinase inhibitory activitycomprising the compound or a salt thereof as claimed in claim 1 as anactive ingredient.
 7. A pharmaceutical composition havinganti-inflammatory activity comprising the compound or a salt thereof asclaimed in claim 1, 2 or 3 as an active ingredient.
 8. Thepharmaceutical composition as claimed in claim 7, wherein saidcomposition is effective for treating or preventing a disease selectedfrom the group consisting of asthma; allergic rhinitis; atopicdermatitis; hives; conjunctivitis; vernal catarrh; chronicarthrorheumatism; systemic lupus erythematosus; psoriasis; diabroticcolitis; systemic inflammatory response syndrome (SIRS); sepsis;polymyositis; dermatomyositis (DM); Polyaritis nodoa (PN); mixedconnective tissue disease (MCTD); Sjoegren's syndrome; and gout.
 9. Apharmaceutical composition having immunosuppressant activity comprisingthe compound or a salt thereof as claimed in claim 1, 2 or 3 as anactive agent.
 10. A pharmaceutical composition for treatment of ischemiacomprising the compound or salt thereof as claimed in claim 1, 2 or 3 asan active agent.
 11. A pharmaceutical composition having anti-tumouractivity comprising the compound or a salt thereof as claimed in claim1, 2 or 3 as an active agent.
 12. (canceled)
 13. A method for treatmentof inflammatory diseases, comprising administering an effective amountof a compound according to claim 1, 2, or
 3. 14. A method of inhibitinganIκB kinase in a subject, comprising administering an effective amountof the compound or a salt thereof as claimed in claim
 1. 15. A method oftreating an inflammatory condition in a subject, comprisingadministering an effective amount of the compound of claim 1, 2, or 3.16. The method of claim 15 wherein said inflammartory condition isselected from the group consisting of asthma; allergic rhinitis; atopicdermatitis; hives; conjunctivitis; vernal catarrh; chronicarthrorheumatism; systemic lupus erythematosus; psoriasis; diabroticcolitis; systemic inflammatory response syndrome (SIRS); sepsis;polymyositis; dermatomyositis (DM); Polyaritis nodoa (PN); mixedconnective tissue disease (MCTD); Sjoegren's syndrome; and gout.
 17. Amethod for suppressing an immune response in a subject, comprisingadministering an effective amount of the compound or salt thereof asclaimed in claim 1, 2, or
 3. 18. A method for treating ischemia in asubject, comprising administering an effective amount of the compound orsalt thereof as claimed in claim 1, 2, or
 3. 19. A method for treatingcancer in a subject, comprising administering an effective amount of thecompound or salt thereof as claimed in claim 1, 2, or 3.